The enrichment of foods with hydrophobic biologically active compounds, including hydrophobic nutraceuticals and fat-soluble vitamins is a promising strategy for promoting health of global populations. However, the desire for reduction of fat content in a healthy diet creates a difficulty in providing the required amounts of essential hydrophobic biologically active compounds, including fat-soluble vitamins and hydrophobic nutraceuticals. Moreover, many hydrophobic biologically active compounds degrade easily during processing, shelf life and digestion. The need for enrichment of food products with hydrophobic biologically active compounds motivates the development of novel technologies for solubilizing, stabilizing and protecting such hydrophobic biologically active compounds using natural food components.
Clear drinks, which are consumed in large quantities, pose a particularly important challenge because of the difficulty of incorporating oil-soluble materials in a clear and stable aqueous dispersion. The ideal vehicle for the task should be nano-sized to maintain transparency and composed of only natural, generally regarded as safe and inexpensive food components, capable of solubilizing and protecting hydrophobic biologically active compounds in aqueous media while retaining sensory qualities, and promoting bioavailability of hydrophobic biologically active compounds. Very few solutions for these challenging requirements have been suggested and none has all the desired attributes. Micro- or nanoemulsions may have excellent transparency (Garti et al., (2004) Physical Chemistry and Chemical Physics 6, 2968; Yaghmur et al., (2004) Physical Chemistry and Chemical Physics 6, 1524), but generally require the presence of a high proportion of synthetic emulsifiers that are not desirable ingredients.
Milk protein-based nanoparticles offer the potential for nanoencapsulation of nutraceuticals (Chen et al., (2006) Trends in Food Sci. and Technology 17, 272; Semo et al., (2007) Food Hydrocolloids 21,936; Subirade et al., (2003) Hemijska Industrija 57, 617), as many of the milk proteins have naturally evolved to deliver nutrients from mother to neonate. β-lactoglobulin was suggested as a suitable vehicle for delivery of hydrophobic biologically active compounds, as it had been shown to bind a variety of lipophilic micronutrients (Wang et al., (1997a) J. Dairy Sci. 80:1047; Wang et al., (1997b) J. Dairy Sci. 80:1054; Zimet and Livney (2009) Food Hydrocolloids 23:1120). It is the major whey protein of ruminant species, (2-3 grams per liter in cow's milk, 18.4 kDa) (Hambling et al., (1992) In P. F. Fox (Ed.), Advanced Dairy Chemistry-1: Proteins p. 141 London & New York: Elsevier Applied Science; Jameson et al., (2002) Int. Dairy J. 12, 319), and its globular structure comprises an 8-stranded antiparallel β-barrel with a 3-turn α-helix on the outer surface (Brownlow et al., (1997) Structure 5, 481). The solvent accessible conical β-barrel, or calyx, forms the main ligand binding site, although evidence suggests that there is a second ligand binding site in a crevice near the α-helix on the external surface of the β-barrel (Kontopidis et al., (2004) Int. Dairy J. 12, 319). Native β-lactoglobulin is stable in acid conditions, and quite resistant to digestion by gastric proteinases (Wang et al., (1997a) J. Dairy Sci. 80, 1047; Wang et al., (1997b) J. Dairy Sci. 80, 1054). However, the main shortcoming of β-lactoglobulin as a nanovehicle for hydrophobic biologically active compounds is that β-lactoglobulin binding sites are solvent-accessible, and thus the ligand is only partly protected from the environment.
U.S. Pat. No. 6,290,974 teaches a food composition comprising a food additive comprising a preformed complex comprising β-lactoglobulin and a lipophilic nutrient selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin K1, cholesterol, and conjugated linoleic acid. In that disclosure the lipophilic nutrient is bound to β-lactoglobulin via one or more amino acid residues. Nowhere in U.S. Pat. No. 6,290,974 is it disclosed, taught or suggested that polysaccharides can be added to β-lactoglobulin-lipophilic nutrient complexes to form clear colloidally stable dispersions of nanoparticles for enhanced protection of the lipophilic nutrient.
Attractive biopolymer interactions mainly occur between positively charged proteins (when the pH<pI) and anionic polysaccharides or negatively charged proteins (when the pH>pI) and cationic polysaccharides. These interactions result in complex formation, and depending on pH, ionic strength and molar ratio of the two biopolymers, may lead to formation of either soluble complexes or to complex coacervation, i.e. associative phase separation. Soluble complexes may be obtained when opposite charges carried by the two macro-ions within a complex are not equal in number. The resulting net charge allows the complex solubilization thanks to the high entropy of the low molecular weight counter ions, as well as to the repulsion between the similarly charged complexes. However, when the opposite charges carried by the two biopolymers neutralize each other, the complexes become insoluble, resulting in coacervation and precipitation (de Kruif et al., (2004) Curr. Opin. Colloid & Interface Sci. 9, 340; Livney, (2007) in N. Garti (Ed.), Delivery and controlled release of bioactives in foods and nutraceuticals. Cambridge, England: Woodhead Publishing Ltd; Schmitt et al., (1998) Critical Reviews in Food Science and Nutrition 38, 689).
Core-shell nanoparticles of chitosan coated with β-lactoglobulin were proposed for delivery of nutraceuticals (Chen and Subirade, (2005) Biomaterials 26, 6041). The particles formed were about 100 nm in size, and were designed to encapsulate negatively charged nutraceuticals, as the cationic chitosan served as the core while β-lactoglobulin was used as the coating material. These nanoparticles were not made of only natural ingredients and preferably included tripolyphosphate as a cross-linker for nano gel-particle formation. In addition, nowhere is it taught or suggested in this reference, that the core-shell β-lactoglobulin-chitosan nanoparticles formed were clear in suspension or that they could be used for hydrophobic nutraceuticals.
Vitamin D is a fat-soluble vitamin of great importance in calcium and phosphate metabolism, i.e. in facilitation of calcium absorption in the intestine, transporting calcium and phosphate to the bones and re-absorption of calcium and phosphate in the kidneys. Vitamin D also takes part in the formation of osteoblasts, in fetal development and in the normal function of the nerve system, the pancreas and the immune system (Eintenmiller, R. R. and Landen, W. O., (1999) In Vitamin analysis for the health and food science Lawson, D. E. M. (Ed.) CRC Press: Boca Raton p. 77). However, because vitamin D is fat-soluble it is practically absent in low-fat and non-fat dairy products, important sources for calcium and phosphate, which are consumed in large quantities, particularly in modern societies. Additionally, solubility of vitamin D and other fat-soluble vitamins in other low-fat and non-fat food products is very low.
Vitamin D has over 40 known metabolites, one of which is vitamin D2. Vitamin D2 originates from plants. It is found in nature in limited amounts, but can be synthesized readily. The vitamin structure contains double bonds that are sensitive to oxidation. Light, air and high temperature induce vitamin isomerization or degradation into inactive products (Eintenmiller, R. R. and Landen, W. O., (1999) In Vitamin analysis for the health and food science CRC Press: Boca Raton p. 77; Bell, A. B. (2005) In Vitamin D Lawson, D. E. M. (Ed.) London, Academic Press p. 1).
Omega-3 polyunsaturated fatty acids are gaining increasing recognition as important nutraceutical lipids, playing significant roles in protecting against cardiovascular diseases, cancer, and inflammation. Moreover, many studies show that consumption of omega-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), positively influences brain development, learning and memory, visual function, and exerts remedial effects on dementia and mental disorders. Not only is DHA poorly soluble in aqueous solutions but because DHA is highly unsaturated, it is also very sensitive to oxidative degradation, leading to off-flavors and odors. Factors affecting oxidation rates include fatty acid composition, storage conditions and physical state.
There remains an unmet need for compositions and methods useful in enriching low-fat and non-fat food products with hydrophobic biologically active compounds. Particularly, there is a need in the art for compositions and methods of providing hydrophobic biologically active compounds as additives with enhanced stability and without compromising bioavailability or sensorial characteristics of foods, such as transparency and flavor.